In a paper by J. B. Griffiths and A. Electricwala entitled "Amplification of Tissue Phasminogen Activator Expression from Epithelial Lines" given at the 7th General Meeting of ESACTR on Advances in Aninmal Cell Technology: Cell Engineering Evaluation and Exploitation, at Baden, Australia, 1985 and published in Develop. biol. Standard., Volume 66 pages 417-422 (S. Karger, Basel 1987), reference is made to a process for achieving expression of tPA from established epithelial cell lines, one derived from human breast tissue (BEB) and another from guinea pig ear keratocytes (GPK). The GPK cell line has been deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institute Pasteur in Paris, France under accession number CNCM I-222.
The enzyme was found to be mainly expressed during the cell growth phase rather than from stationary culture cells, and production involved a two-step operation with initial growth to about 70% confluency in the presence of serum followed by a change to serum-free conditions for the final period of growth, after which the enzyme was harvested.
The paper discusses various approaches to increase the enzyme yield. The addition of mitogenic lectins was reported to increase the enzyme yield 15-20 fold.
In a typical enzyme production process based on this approach, cells are inoculated into roller bottles and incubated at 36.5.degree. C. for 72 hours in culture medium containing animal serum, resulting in growth to about 70% confluency. At the end of the 72 hours the growth medium is poured off, the cell washed twice and the medium replaced with half the original volume of culture medium containing no serum but with 50 microgarms/ml of a lectin, the most effective of which was found to be concanavalin A. Experiments showed this to be the optimum concentration of concanavalin A. The cultures are then incubated in the presence of lectin for a further 48 hours at 36.5.degree. C. after which supernatants (containing enzyme) are harvested and the cultures discarded.